Exposure to short wave ultraviolet light has been demonstrated to be the causal factor in nonmelanoma skin cancers and a strong risk factor in melanomas. While human cells only use nucleotide excision repair to repair the UV-induced dipyrimidine DNA photoproducts, other organisms initiate the base excision repair pathway by DNA glycosylases that catalyze incision at the 5'base of pyrimidine dimers. Developing an understanding of the function of enzymes is critical, since T4 pyrimidine dimer glycosylase (T4-Pdg) is being used in human clinical trials. Although topical delivery of wild-type T4-Pdg on xeroderma pigmentosum patients has demonstrated efficacy in cancer reduction, all investigations to date using wild-type mammalian cells reveal that T4-Pdg results in decreased, rather than increased survival after UV. It is hypothesized that the ability of T4-Pdg to incise all dimer sites within DNA domains leads to cytotoxic double-strand breaks where dimers are in close proximity in complementary strands. Thus, it is hypothesized that forms of T4-Pdg that have lost the ability to incise dimers in clusters will enhance repair and decrease mutagenesis without creating cytotoxic double-strand breaks. To accomplish this goal, it is proposed to engineer T4-Pdg to be less efficient in the precatalytic steps of DNA bending and nucleotide flipping, with the net result being a decrease in the ability of these altered enzymes to form a Michaelis complex and incise dimers in clusters. These studies will be guided by our recent determination of the cocrystal structure of T4-Pdg covalently trapped as a reduced imine intermediate at an abasic site in duplex DNA. This structure reveals key amino acids necessary for achieving an active complex, and these data have led to a series of hypotheses that implicate at least three different portions of the enzyme in this process. Using the knowledge derived from the cocrystal structure, and given the challenges of enhancing dimer repair in wild-type mammalian cells, Specific Aims are proposed to 1) biochemically characterize mutants of T4-Pdg in their ability to carry out bending, flipping, catalysis, and clustered incisions in vitro;2) express control and mutant T4-Pdgs in keratinocytes to determine effects on double-strand break formation, survival, and mutagenesis;and 3) activate base excision repair of UV-photoproducts in mitochondria and determine the role of dimers in cytotoxicity.